Lipophilic drug delivery vehicle and methods of use thereof

ABSTRACT

The invention provides compositions and methods for delivery of a bioactive agent to an individual. Delivery vehicles are provided that include a bioactive agent in disc shaped particles that include one or more lipid binding polypeptides circumscribing the perimeter of a lipid bilayer in which the bioactive agent is localized. Chimeric lipid binding polypeptides are also provided and may be used to add additional functional properties to the delivery particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 13/585,598, filed Aug. 14, 2012, which is a continuation ofU.S. patent application Ser. No. 12/815,058, filed Jun. 14, 2010, nowU.S. Pat. No. 8,268,357, which is a divisional of U.S. patentapplication Ser. No. 10/778,640, filed Feb. 13, 2004, now U.S. Pat. No.7,824,709, which claims the benefit of U.S. Provisional Application Nos.60/447,508, filed Feb. 14, 2003, and 60/508,035, filed Oct. 1, 2003, andwhich are hereby incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant no.HL-064159 from the National Institutes of Health. The government hascertain rights in this invention.

FIELD OF THE INVENTION

This application relates to compositions and methods for delivery ofbioactive agents. In particular, the application relates to bioactiveagent delivery particles that include a lipid binding polypeptide, alipid bilayer, and a bioactive agent.

BACKGROUND OF THE INVENTION

Bioactive substances such as therapeutic agents, vaccine immunogens, andnutrients often cannot be administered in pure form, but must beincorporated into biocompatible formulations that enhance solubility ofthe bioactive material and package it in a suitable form to achieveoptimal beneficial effects while minimizing undesirable side effects.Efficient delivery of bioactive agents is often hindered by a shortclearance time of an agent in the body, inefficient targeting to a siteof action, or the nature of the bioactive agent itself, for example,poor solubility in aqueous media or hydrophobicity. Thus, manyformulation strategies have been developed to improve delivery,including controlled release formulations, emulsions, and liposomalpreparations.

Liposomal pharmaceutical delivery systems have been described. Liposomesare completely closed, spherical lipid bilayer membranes containing anentrapped aqueous volume. The lipid bilayer includes two lipidmonolayers composed of lipids having a hydrophobic tail region and ahydrophilic head region. The structure of the membrane bilayer is suchthat the hydrophobic, nonpolar tails of the lipid molecules orienttoward the center of the bilayer while the hydrophilic heads orienttoward the aqueous phases both on the exterior and the interior of theliposome. The aqueous, hydrophilic core region of a liposome may includea dissolved bioactive substance.

Delivery of pharmaceutically useful hydrophobic substances is oftenparticularly problematic because they are insoluble or poorly soluble inan aqueous environment. For hydrophobic compounds used aspharmaceuticals, direct injection may be impossible or highlyproblematic, resulting in such dangerous conditions as hemolysis,phlebitis, hypersensitivity, organ failure, and/or death. There is aneed for improved formulations for hydrophobic bioactive substances thatwill promote stability in an aqueous environment and allow efficientdelivery of such substances to a desired site of action.

BRIEF SUMMARY OF THE INVENTION

The invention provides compositions and methods for delivery of abioactive agent to an individual.

In one aspect, the invention provides a bioactive agent deliveryparticle that includes a lipid binding polypeptide, a lipid bilayer withan interior that includes a hydrophobic region, and a bioactive agentassociated with the hydrophobic region of the lipid bilayer. Bioactiveagent delivery particles generally do not include a hydrophilic oraqueous core.

Bioactive agent delivery particles include one or more bioactive agentsthat include at least one hydrophobic region and are incorporated into,or associated with, the hydrophobic interior of the lipid bilayer. Thehydrophobic region(s) of a bioactive agent are generally associated withhydrophobic surfaces in the interior of the lipid bilayer, e.g., fattyacyl chains. In one embodiment, the bioactive agent is amphotericin B(AmB). In another embodiment, the bioactive agent is camptothecin.

Particles are typically disc shaped, with a diameter in the range ofabout 7 to about 29 nm.

Bioactive agent delivery particles include bilayer-forming lipids, forexample phospholipids. In some embodiments, a bioactive agent deliveryparticle includes both bilayer-forming and non-bilayer-forming lipids.In some embodiments, the lipid bilayer of a bioactive agent deliveryparticle includes phospholipids. In one embodiment, the phospholipidsincorporated into a delivery particle includedimyristoylphosphatidylcholine (DMPC) anddimyristoylphosphatidylglycerol (DMPG). In one embodiment, the lipidbilayer includes DMPC and DMPG in a 7:3 molar ratio.

In a preferred embodiment, the lipid binding polypeptide is anapolipoprotein. The predominant interaction between lipid bindingpolypeptides, e.g., apolipoprotein molecules, and the lipid bilayer isgenerally a hydrophobic interaction between residues on a hydrophobicface of an amphipathic structure, e.g., an α-helix of the lipid bindingpolypeptide and fatty acyl chains of lipids on an exterior surface atthe perimeter of the particle. Particles of the invention may includeexchangeable and/or non-exchangeable apolipoproteins. In one embodiment,the lipid binding polypeptide is Apolipoprotein A-I (ApoA-I).

In some embodiments, particles are provided that include lipid bindingpolypeptide molecules, e.g., apolipoprotein molecules, that have beenmodified to increase stability of the particle. In one embodiment, themodification includes introduction of cysteine residues to formintramolecular and/or intermolecular disulfide bonds.

In another embodiment, particles are provided that include a chimericlipid binding polypeptide molecule, e.g., a chimeric apolipoproteinmolecule, with one or more bound functional moieties, for example one ormore targeting moieties and/or one or more moieties having a desiredbiological activity, e.g., antimicrobial activity, which may augment orwork in synergy with the activity of a bioactive agent incorporated intothe delivery particle.

In another aspect, a pharmaceutical composition is provided thatincludes a bioactive agent delivery particle in a pharmaceuticallyacceptable carrier. A method for administering a bioactive agent to anindividual is also provided, which includes administering apharmaceutical composition containing bioactive agent delivery particlesin a pharmaceutically acceptable carrier to the individual. In someembodiments, a therapeutically effective amount of the bioactive agentis administered in a pharmaceutically acceptable carrier. In someembodiments, administration is parenteral, for example intravenous,intramuscular, transmucosal, or intrathecal. In other embodiments,particles are administered as an aerosol. In some embodiments, thebioactive agent is formulated for controlled release. In one embodiment,a method is provided for treating a fungal infection in an individual,including administering a anti-fungal agent, for example, AmB,incorporated into bioactive agent delivery particles of the invention,often in a therapeutically effective amount in a pharmaceuticallyacceptable carrier. In another embodiment, a method is provided fortreating a tumor in an individual, including administering an anti-tumoragent, for example, camptothecin, incorporated into bioactive agentdelivery particles of the invention, often in a therapeuticallyeffective amount in a pharmaceutically acceptable carrier. In oneembodiment, the bioactive agent delivery particles include a lipidbinding polypeptide with an attached vasoactive intestinal peptidetargeting moiety, and the tumor is a breast tumor.

In a still further aspect, processes are provided for formulatingbioactive agent delivery particles as described above. In oneembodiment, the formulation process includes contacting a mixture thatincludes bilayer-forming lipids and a bioactive agent to form a lipidvesicle-bioactive agent mixture, and contacting the lipidvesicle-bioactive agent mixture with a lipid binding polypeptide. Inanother embodiment, the formulation process includes formation of adispersion of pre-formed bilayer-containing lipid vesicles to which abioactive agent, dissolved in an appropriate solvent, is added.Appropriate solvents for solubilizing a bioactive agent for thisprocedure include solvents with polar or hydrophilic character that arecapable of solubilizing a bioactive agent to be incorporated into adelivery particle of the invention. Examples of suitable solventsinclude, but are not limited to, dimethylsulfoxide (DMSO) anddimethylformamide. To the vesicle/bioactive agent mixture, lipid bindingpolypeptides are added, followed by incubation, sonication, or both. Inone embodiment, the bioactive agent incorporated into a deliveryparticle by any of the above processes is amphotericin B. In oneembodiment, the amphotericin B is solubilized in DMSO. In anotherembodiment, the bioactive agent is camptothecin. In one embodiment, thecamptothecin is solubilized in DMSO.

The invention includes bioactive agent delivery particles preparedaccording to any of the processes described above, and pharmaceuticalcompositions including particles prepared according to any of the aboveprocesses and a pharmaceutically acceptable carrier.

In another aspect, the invention provides kits including any of thebioactive agent delivery particles or pharmaceutical compositionsdescribed above, or delivery particles prepared by any of the abovemethods, and/or reagents for formulating the particles and/orinstructions for use in a method for administering a bioactive agent toan individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a UV/Visible absorbance spectrum, from 250-450 nm, ofApoA-I-phospholipid particles without a bioactive agent, prepared as inExample 1.

FIG. 2 depicts a UV/Visible absorbance spectrum, from 250-450 nm, ofApoA-I-phospholipid-AmB particles, prepared as in Example 1.

FIG. 3 depicts a plot of fraction number versus protein concentrationfor ApoA-I-phospholipid-AmB particles after density gradientultracentrifugation. Particles were prepared as described in Example 2and adjusted to 1.3 g/ml density by the addition of KBr. The solutionwas centrifuged in a discontinuous gradient for 5 hours at 275,000×g at10° C. Following centrifugation, the tube contents were fractionatedfrom the top and the protein content in each fraction determined.

FIG. 4 depicts a native polyacrylamide gel electrophoresis (PAGE)analysis of ApoA-I-phospholipid particles, on a 4-20% acrylamidegradient slab gel. Particles were prepared with ApoA-I and two differentlipid preparations, DMPC/DMPG or palmitoyloleylphosphatidylcholine(POPC). The gel was stained with Coomassie Blue. Lane 1: ApoA-I POPCparticles; Lane 2: ApoA-I-POPC-AmB particles; Lane 3:ApoA-I-DMPC/DMPG-AmB particles. The relative migration of size standardsis shown on the left.

FIG. 5 depicts a comparison of effects of different storage conditionson the size and structural integrity of Apoliprotein E N-terminal domain(ApoE3NT)-DMPC/DMPG-AmB particle stability. Particles were isolated bydensity ultracentrifugation and then subjected to electrophoresis on anative PAGE 4-20% gradient slab gel. The gel was stained with AmidoBlack. Lane 1: particles stored in phosphate buffer at 4° C. for 24hours; Lane 2: particles stored in phosphate buffer at −20° C. for 24hours; Lane 3: particles lyophilized and frozen at −80° C. for 24 hours,and then redissolved in H₂O. The relative migration of size standards isshown on the left.

FIGS. 6A and 6B schematically illustrate the shape and molecularorganization of a bioactive agent delivery particle.

FIGS. 7A, 7B, and 7C schematically illustrate chimeric lipid bindingpolypeptides and their incorporation into a bioactive agent deliveryparticle. The chimeric proteins may include a targeting moiety (FIG. 7A)or a moiety with a desired biological activity (FIG. 7B). FIG. 7Cschematically illustrates incorporation of the chimeric polypeptidesshown in FIGS. 7A and 7B into a bioactive agent delivery particle.

FIG. 8 graphically depicts antifungal activity of AmB-containingbioactive agent delivery particles against Saccharomyces cerevisiae (S.cerevisiae) in culture, as described in Example 2.

FIG. 9 is a freeze fracture electron micrograph of AmB-containingbioactive agent delivery particles, prepared as described in Example 10.

FIG. 10 shows a comparison between the ability of ApoA-1-DMPC/DMPG-AmBparticles and AmBisome® to inhibit growth of S. cerevisiae, as describedin Example 8.

FIG. 11 shows fluorescence spectral comparison between camptothecinsolubilized in SDS (FIG. 11, line A) and camptothecin-containingbioactive agent delivery particles (FIG. 11, line B), as described inExample 9.

FIG. 12 depicts a UV/visible spectral comparison of AmB incorporationinto lipid particles prepared as described in Example 7 (FIG. 12, lineA) and bioactive agent delivery particles prepared as described inExample 6 (FIG. 12, line B).

FIG. 13 is an illustration of an embodiment of a bioactive agentdelivery particle preparation procedure.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for delivery of abioactive agent to an individual. Delivery vehicles are provided in theform of a bioactive agent incorporated into a particle that includes alipid binding polypeptide and a lipid bilayer. The interior of theparticle includes a hydrophobic region of the lipid bilayer thatincludes hydrophobic portions of lipid molecules, e.g., fatty acylchains of lipids, in contrast to liposomes, which include a whollyenclosed aqueous interior surrounded by lipid hydrophilic surfaces of abilayer. The hydrophobic nature of the interior of a particle of theinvention permits incorporation of hydrophobic molecules, for example,by intercalation between lipid molecules in the bilayer or sequestrationinto the hydrophobic region between leaflets of the bilayer. A bioactiveagent that includes at least one hydrophobic region may be incorporatedinto the hydrophobic interior of the particle. As used herein,“incorporation” of a bioactive agent into the hydrophobic region of alipid bilayer refers to solubilization into or association with ahydrophobic region or hydrophobic portions of lipid molecules of thebilayer, e.g., fatty acyl chains of lipids that form the bilayer, orintercalation with the fatty acyl chains.

The particles are generally disc shaped, with a diameter in the range ofabout 7 to 29 nm, as determined by native pore limiting gradient gelelectrophoresis, in comparison with standards of known Stokes' diameter,as described, for example, in Blanche et al. (1981) Biochim. Biophys.Acta. 665(3):408-19. In some embodiments, the particles are stable insolution and may be lyophilized for long term storage, followed byreconstitution in aqueous solution. The lipid binding polypeptidecomponent defines the boundary of the discoidal bilayer and providesstructure and stability to the particles.

Chimeric lipid binding polypeptide molecules (e.g., apolipoproteinmolecules) are also provided and may be used to incorporate variousadditional functional properties into the delivery particles of theinvention.

The particles may be administered to an individual to deliver abioactive agent to the individual.

Bioactive Agent Delivery Particles

The invention provides a “particle” (also termed “delivery particle” or“bioactive agent delivery particle” herein) that includes one or moretypes of lipid binding polypeptide, a lipid bilayer comprising one ormore types of bilayer-forming lipid, and one or more bioactive agents.In some embodiments, a delivery particle also includes one or more typesof non-bilayer-forming lipid. Compositions including the particles arealso provided. In one embodiment, a pharmaceutical composition isprovided that includes delivery particles and a pharmaceuticallyacceptable carrier.

The interior of a particle includes a hydrophobic region (e.g.,comprised of lipid fatty acyl chains). Particles of the inventiontypically do not comprise a hydrophilic or aqueous core. The particlesare generally disc shaped, having a flat, discoidal, roughly circularlipid bilayer circumscribed by amphipathic α-helices and/or β-sheets ofthe lipid binding polypeptides, which are associated with hydrophobicsurfaces of the bilayer around the periphery of the disc. Anillustrative example of a disc shaped bioactive agent delivery particleof the invention is schematically depicted in FIG. 6.

Typically, the diameter of a disc shaped delivery particle is about 7 toabout 29 nm, often about 10 to about 25 nm, often about 15 to about 20nm. “Diameter” refers to the diameter of one of the roughly circularshaped faces of the disc.

Lipid Binding Polypeptides

As used herein, a “lipid binding polypeptide” refers to any synthetic ornaturally occurring peptide or protein that forms a stable interactionwith lipid surfaces and can function to stabilize the lipid bilayer of aparticle of the invention. Particles may include one or more types oflipid binding polypeptides, i.e., the lipid binding polypeptides in asingle particle may be identical or may be composed of two or moredifferent polypeptide sequences. The lipid binding polypeptidescircumscribe the periphery of the particle.

In some embodiments, lipid binding polypeptides useful for producingdelivery particles in accordance with the invention include proteinshaving an amino acid sequence of a naturally occurring protein, or afragment, natural variant, isoform, analog, or chimeric form thereof,proteins having a non-naturally occurring sequence, and proteins orpeptides of any length that possess lipid binding properties consistentwith known apolipoproteins, and may be purified from natural sources,produced recombinantly, or produced synthetically. An analog of anaturally-occurring protein may be used. A lipid binding polypeptide mayinclude one or more non-natural amino acids (e.g., D-amino acids), aminoacid analogs, or a peptidomimetic structure, in which the peptide bondis replaced by a structure more resistant to metabolic degradation, orindividual amino acids are replaced by analogous structures.

In a preferred embodiment, the lipid binding polypeptide is anapolipoprotein. Any apolipoprotein or fragment or analog thereof may beused that is capable of associating with a lipid bilayer to form a discshaped particle. Particles may include exchangeable, non-exchangeable,or a mixture of exchangeable and non-exchangeable apolipoproteinmolecules.

Apolipoproteins generally possess a class A amphipathic α-helixstructural motif (Segrest et al. (1994) Adv. Protein Chem. 45:303-369),and/or a β-sheet motif. Apolipoproteins generally include a high contentof α-helix secondary structure with the ability to bind to hydrophobicsurfaces. A characteristic feature of these proteins is their ability tointeract with certain lipid bilayer vesicles and to transform them intodisc-shaped complexes (for a review, see Narayanaswami and Ryan (2000)Biochimica et Biophysica Acta 1483:15-36). Upon contact with lipids, theprotein undergoes a conformational change, adapting its structure toaccommodate lipid interaction.

Generally, the predominant interaction between apolipoproteins and thelipid bilayer in a particle is through a hydrophobic interaction betweenresidues on the hydrophobic faces of amphipathic α-helices ofapolipoprotein molecules and hydrophobic surfaces of lipids, forexample, phospholipid fatty acyl chains, at the edge of the bilayer atthe periphery of the bioactive agent delivery particle. An amphipathicα-helix of an apolipoprotein molecule includes both a hydrophobicsurface in contact with a hydrophobic surface of the lipid bilayer atthe periphery of the particle, and a hydrophilic surface facing theexterior of the particle and in contact with the aqueous environmentwhen the particle is suspended in aqueous medium. In some embodiments,an apolipoprotein may include an amphipathic β-sheet structure whereinhydrophobic residues of the β-sheet interact with lipid hydrophobicsurfaces at the periphery of the disc.

A bioactive agent delivery particle often comprises about 1 to about 10molecules of one or more types of apolipoprotein per particle. Theamount of amphipathic α-helix contributed by the apolipoproteins in aparticle is generally sufficient to cover the otherwise exposedhydrophobic surface of the lipid molecules located at the edge of thedisc shaped lipid bilayer (i.e., the periphery of the particle).

In one embodiment in which the apolipoprotein is human apolipoproteinA-I (ApoA-I) and the lipid bilayer includespalmitoyloleoylphosphatidylcholine, a particle comprises 2 ApoA-1molecules in a ratio of about 80 molecules of phospholipid to about 1molecule of ApoA-I.

Examples of apolipoproteins which may be used for formation of thedelivery particles of the invention include, but are not limited to,ApoA-I, apolipoprotein E (ApoE), and apolipophorin III (ApoIII),apolipoprotein A-IV (ApoA-IV), apolipoprotein A-V (ApoA-V),apolipoprotein C-I (ApoC-I), apolipoprotein C-II (ApoC-II),apolipoprotein C-III (ApoC-III), apolipoprotein D (ApoD), apolipoproteinA-II (ApoA-II), apolipoprotein B-100 (ApoB-100), apolipoprotein J(ApoJ), apolipoprotein H (ApoH), or fragments, natural variants,isoforms, analogs, or chimeric forms thereof. In some embodiments, theapolipoprotein is human ApoA-I. In other embodiments, the apolipoproteinis the C-terminal or N-terminal domain of apolipoprotein E3, or isoformsthereof. In some embodiments, the apolipoprotein includes a functionalmoiety that has been attached either synthetically or recombinantly,such as a targeting moiety or a moiety having biological activity, thatis not intrinsic to the apolipoprotein (see, e.g., FIG. 7).

In some embodiments, an exchangeable apolipoprotein is used. An“exchangeable apolipoprotein” may be displaced from a preformeddiscoidal particle of the invention by another protein or peptide withlipid binding affinity, without destroying the integrity of theparticle. Exchangeable apolipoproteins include synthetic or naturalpeptides or proteins capable of forming a stable binding interactionwith lipids. More than a dozen unique exchangeable apolipoproteins havebeen identified in both vertebrates and invertebrates (see, e.g.,Narayanaswami and Ryan, supra).

In some embodiments, a non-exchangeable apolipoprotein is used. As usedherein, “non-exchangeable apolipoprotein” refers to a protein or peptidethat forms a stable interaction with lipid surfaces and can function tostabilize the phospholipid bilayer of particles of the invention, butcannot be removed from the surface of the particle without destroyingthe intrinsic structure of the particle.

Bioactive Agents

The delivery particles include one or more bioactive agents. As usedherein, “bioactive agent” refers to any compound or composition havingbiological, including therapeutic or diagnostic, activity. A bioactiveagent may be a pharmaceutical agent, drug, compound, or composition thatis useful in medical treatment, diagnosis, or prophylaxis.

Bioactive agents incorporated into delivery particles as describedherein generally include at least one hydrophobic (e.g., lipophilic)region capable of associating with or integrating into the hydrophobicportion of a lipid bilayer. In some embodiments, at least a portion ofthe bioactive agent is intercalated between lipid molecules in theinterior of the delivery particle. Examples of bioactive agents that maybe incorporated into delivery particles in accordance with the inventioninclude, but are not limited to, antibiotic or antimicrobial (e.g.,antibacterial, antifungal, and antiviral) agents, antimetabolic agents,antineoplastic agents, steroids, peptides, proteins, such as, forexample, cell receptor proteins, enzymes, hormones, andneurotransmitters, radiolabels such as radioisotopes andradioisotope-labeled compounds, fluorescent compounds, anesthetics,bioactive lipids, anticancer agents, anti-inflammatory agents,nutrients, antigens, pesticides, insecticides, herbicides, or aphotosensitizing agent used in photodynamic therapy. In one embodiment,the bioactive agent is the anti-fungal agent AmB. In other embodiments,the bioactive agent is camptothecin, all-trans retinoic acid, annamycin,nystatin, paclitaxel, docetaxel, or etiopurpurins. Bioactive agents thatinclude at least one hydrophobic region are known in the art andinclude, but are not limited to, ibuprofen, diazepam, griseofulvin,cyclosporin, cortisone, proleukin, etoposide, taxane, α-tocopherol,Vitamin E, Vitamin A, and lipopolysaccharides. See, for example,Kagkadis et al. (1996) PDA J Pharm Sci Tech 50(5):317-323; Dardel (1976)Anaesth Scand 20:221-24; Sweetana and Akers (1996) PDA J Pharm Sci Tech50(5):330-342; U.S. Pat. No. 6,458,373.

In some embodiments, a bioactive agent incorporated into a deliveryparticle of the invention is a non-polypeptide. In some embodiments, foradministration to an individual, a bioactive agent and the deliveryparticle that includes the bioactive agent are substantiallynonimmunogenic when administered to an individual.

Lipid Bilayer

Particles of the invention include a lipid bilayer, with the generallycircular faces of the disc comprising polar head groups facing away fromthe interior of the particle, and the interior of the particle (i.e.,the space between the circular faces) comprising a hydrophobic region ofthe lipid bilayer that contains hydrophobic portions of bilayer-forminglipid(s) and other lipid components, if present. Hydrophobic surfaces ofthe lipid molecules at the edge of the bilayer (the surface at theperiphery of the bioactive agent delivery particle) contact the lipidbinding polypeptides of the particles, as discussed above. Particles mayinclude one or more types of bilayer-forming lipids, or a mixture of oneor more types of bilayer-forming and one or more types ofnon-bilayer-forming lipids. As used herein, “lipid” refers to asubstance of biological or synthetic origin that is soluble or partiallysoluble in organic solvents or which partitions into a hydrophobicenvironment when present in aqueous phase.

Any bilayer-forming lipid that is capable of associating with a lipidbinding polypeptide to form a disc shaped structure may be used inaccordance with the invention. As used herein, “bilayer-forming lipid”refers to a lipid that is capable of forming a lipid bilayer with ahydrophobic interior and a hydrophilic exterior. Bilayer-forming lipidsinclude, but are not limited to, phospholipids, sphingolipids,glycolipids, alkylphospholipids, ether lipids, and plasmalogens. Onetype of bilayer-forming lipid may be used or a mixture of two or moretypes. In some embodiments, the lipid bilayer includes phospholipids.Examples of suitable phospholipids include, but are not limited to,DMPC, DMPG, POPC, dipalmitoylphosphatidylcholine (DPPC),dipalmitoylphosphatidylserine (DPPS), cardiolipin,dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol(DSPG), egg yolk phosphatidylcholine (egg PC), soy beanphosphatidylcholine, phosphatidylinositol, phosphatidic acid,sphingomyelin, and cationic phospholipids. Examples of other suitablebilayer-forming lipids include cationic lipids and glycolipids. In oneembodiment, the particles include a phospholipid bilayer of DMPC andDMPG, often in a molar ratio of about 7:3. In another embodiment, theparticles include a phospholipid bilayer of POPC. In some embodiments,mixtures of bilayer-forming lipids may be used in molar ratios of any ofat least about 1:100, 1:50, 1:20, 1:10, 1:5, 3:7, 1:2, or 1:1.

Particles may also include lipids that are not bilayer-forming lipids.Such lipids include, but are not limited to, cholesterol, cardiolipin,phosphatidylethanolamine (this lipid may form bilayers under certaincircumstances), oxysterols, plant sterols, ergosterol, sitosterol,cationic lipids, cerebrosides, sphingosine, ceramide, diacylglycerol,monoacylglycerol, triacylglycerol, gangliosides, ether lipids,alkylphospholipids, plasmalogens, prostaglandins, and lysophospholipids.In some embodiments, a lipid used for preparation of a delivery particlemay include one or more bound functional moieties, such as targetingmoieties, bioactive agents, or tags for purification or detection.

Chimeric Lipid Binding Polypeptides

The invention provides chimeric lipid binding polypeptides, which may beused to prepare the delivery particles described above. A chimeric lipidbinding polypeptide may include one or more attached “functionalmoieties,” such as for example, one or more targeting moieties, a moietyhaving a desired biological activity, an affinity tag to assist withpurification, and/or a reporter molecule for characterization orlocalization studies. An attached moiety with biological activity mayhave an activity that is capable of augmenting and/or synergizing withthe biological activity of a bioactive agent incorporated into thedelivery particle. For example, a moiety with biological activity mayhave antimicrobial (for example, antifungal, antibacterial,anti-protozoal, bacteriostatic, fungistatic, or antiviral) activity. Inone embodiment, an attached functional moiety of a chimeric lipidbinding polypeptide is not in contact with hydrophobic surfaces of thelipid bilayer when the lipid binding polypeptide is incorporated into abioactive agent delivery particle. In another embodiment, an attachedfunctional moiety is in contact with hydrophobic surfaces of the lipidbilayer when the lipid binding polypeptide is incorporated into abioactive agent delivery particle. In some embodiments, a functionalmoiety of a chimeric lipid binding polypeptide may be intrinsic to anatural protein. In some embodiments, a chimeric lipid bindingpolypeptide includes a ligand or sequence recognized by or capable ofinteraction with a cell surface receptor or other cell surface moiety.

In some embodiments, a chimeric lipid binding polypeptide is a chimericapolipoprotein. In one embodiment, a chimeric apolipoprotein includes atargeting moiety that is not intrinsic to the native apolipoprotein,such as for example, S. cerevisiae α-mating factor peptide, folic acid,transferrin, or lactoferrin. In another embodiment, a chimericapolipoprotein includes a moiety with a desired biological activity thataugments and/or synergizes with the activity of a bioactive agentincorporated into the delivery particle, such as for example,histatin-5, magainin peptide, mellitin, defensin, colicin, N-terminallactoferrin peptide, echinocandin, hepcidin, bactenicin, orcyclosporine. In one embodiment, a chimeric lipid binding polypeptidemay include a functional moiety intrinsic to an apolipoprotein. Oneexample of an apolipoprotein intrinsic functional moiety is theintrinsic targeting moiety formed approximately by amino acids 130-150of human ApoE, which comprises the receptor binding region recognized bymembers of the low density lipoprotein receptor family. Other examplesof apolipoprotein intrinsic functional moieties include the region ofApoB-100 that interacts with the low density lipoprotein receptor andthe region of ApoA-I that interacts with scavenger receptor type B 1. Inother embodiments, a functional moiety may be added synthetically orrecombinantly to produce a chimeric lipid binding polypeptide.

As used herein, “chimeric” refers to two or more molecules that arecapable of existing separately and are joined together to form a singlemolecule having the desired functionality of all of its constituentmolecules. The constituent molecules of a chimeric molecule may bejoined synthetically by chemical conjugation or, where the constituentmolecules are all polypeptides or analogs thereof, polynucleotidesencoding the polypeptides may be fused together recombinantly such thata single continuous polypeptide is expressed. Such a chimeric moleculeis termed a fusion protein. A “fusion protein” is a chimeric molecule inwhich the constituent molecules are all polypeptides and are attached(fused) to each other such that the chimeric molecule forms a continuoussingle chain. The various constituents can be directly attached to eachother or can be coupled through one or more linkers.

A “linker” or “spacer” as used herein in reference to a chimericmolecule refers to any molecule that links or joins the constituentmolecules of the chimeric molecule. A number of linker molecules arecommercially available, for example from Pierce Chemical Company,Rockford Ill. Suitable linkers are well known to those of skill in theart and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, or peptide linkers. Wherethe chimeric molecule is a fusion protein, the linker may be a peptidethat joins the proteins comprising a fusion protein. Although a spacergenerally has no specific biological activity other than to join theproteins or to preserve some minimum distance or other spatialrelationship between them, the constituent amino acids of a peptidespacer may be selected to influence some property of the molecule suchas the folding, net charge, or hydrophobicity.

In some embodiments, a chimeric lipid binding polypeptide, such as achimeric apolipoprotein, is prepared by chemically conjugating the lipidbinding polypeptide molecule and the functional moiety to be attached.Means of chemically conjugating molecules are well known to those ofskill in the art. Such means will vary according to the structure of themoiety to be attached, but will be readily ascertainable to those ofskill in the art.

Polypeptides typically contain a variety of functional groups, e.g.,carboxylic acid (—COOH), free amino (—NH₂), or sulfhydryl (—SH) groups,that are available for reaction with a suitable functional group on thefunctional moiety or on a linker to bind the moiety thereto. Afunctional moiety may be attached at the N-terminus, the C-terminus, orto a functional group on an interior residue (i.e., a residue at aposition intermediate between the N- and C-termini) of an apolipoproteinmolecule. Alternatively, the apolipoprotein and/or the moiety to betagged can be derivatized to expose or attach additional reactivefunctional groups.

In some embodiments, lipid binding polypeptide fusion proteins thatinclude a polypeptide functional moiety are synthesized usingrecombinant expression systems. Typically, this involves creating anucleic acid (e.g., DNA) sequence that encodes the lipid bindingpolypeptide and the functional moiety such that the two polypeptideswill be in frame when expressed, placing the DNA under the control of apromoter, expressing the protein in a host cell, and isolating theexpressed protein.

Lipid binding polypeptide sequences and sequences encoding functionalmoieties as described herein may be cloned, or amplified by in vitromethods, such as, for example, the polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), or the self-sustained sequence replication system (SSR). Awide variety of cloning and in vitro amplification methodologies arewell known to persons of skill Examples of techniques sufficient todirect persons of skill through in vitro amplification methods are foundfor example, in Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCRProtocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson(Oct. 1, 1990) C & EN 36-47; The Journal Of NIH Research (1991) 3:81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelliet al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989)J. Clin. Chem., 35: 1826; Landegren et al., (1988) Science, 241:1077-1080; Van Brunt (1990) Biotechnology, 8: 291-294; Wu and Wallace,(1989) Gene, 4: 560; and Barringer et al. (1990) Gene, 89: 117.

In addition, DNA encoding desired fusion protein sequences may beprepared synthetically using methods that are well known to those ofskill in the art, including, for example, direct chemical synthesis bymethods such as the phosphotriester method of Narang et al. (1979) Meth.Enzymol. 68: 90-99, the phosphodiester method of Brown et al. 1979)Meth. Enzymol. 68: 109-151, the diethylphosphoramidite method ofBeaucage et al. (1981) Tetra. Lett., 22: 1859-1862, or the solid supportmethod of U.S. Pat. No. 4,458,066.

A nucleic acid encoding a chimeric lipid binding polypeptide fusionpolypeptide can be incorporated into a recombinant expression vector ina form suitable for expression in a host cell. As used herein, an“expression vector” is a nucleic acid which, when introduced into anappropriate host cell, can be transcribed and translated into apolypeptide. The vector may also include regulatory sequences such aspromoters, enhancers, or other expression control elements (e.g.,polyadenylation signals). Such regulatory sequences are known to thoseskilled in the art (see, e.g., Goeddel (1990) Gene ExpressionTechnology: Meth. Enzymol. 185, Academic Press, San Diego, Calif.;Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology 152 Academic Press, Inc., San Diego, Calif.; Sambrook et al.(1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY, etc.).

In some embodiments, a recombinant expression vector for production of achimeric lipid binding polypeptide is a plasmid or cosmid. In otherembodiments, the expression vector is a virus, or portion thereof, thatallows for expression of a protein encoded by the nucleic acidintroduced into the viral nucleic acid. For example, replicationdefective retroviruses, adenoviruses and adeno-associated viruses can beused. Expression vectors may be derived from bacteriophage, includingall DNA and RNA phage (e.g., cosmids), or viral vectors derived from alleukaryotic viruses, such as baculoviruses and retroviruses, adenovirusesand adeno-associated viruses, Herpes viruses, Vaccinia viruses and allsingle-stranded, double-stranded, and partially double-stranded DNAviruses, all positive and negative stranded RNA viruses, and replicationdefective retroviruses. Another example of an expression vector is ayeast artificial chromosome (YAC), which contains both a centromere andtwo telomeres, allowing YACs to replicate as small linear chromosomes.Another example is a bacterial artificial chromosome (BAC).

The chimeric lipid binding polypeptide fusion proteins of this inventioncan be expressed in a host cell. As used herein, the term “host cell”refers to any cell or cell line into which a recombinant expressionvector for production of a chimeric apolipoprotein fusion protein, asdescribed above, may be transfected for expression. Host cells includeprogeny of a single host cell, and the progeny may not necessarily becompletely identical (in morphology or in total genomic DNA complement)to the original parent cell due to natural, accidental, or deliberatemutation. A host cell includes cells transfected or transformed in vivowith an expression vector as described above. Suitable host cellsinclude, but are not limited to, bacterial cells (e.g. E. coli), fungalcells (e.g., S. cerevisiae), invertebrate cells (e.g. insect cells suchas SF9 cells), and vertebrate cells including mammalian cells.

An expression vector encoding a chimeric lipid binding polypeptidefusion protein can be transfected into a host cell using standardtechniques. “Transfection” or “transformation” refers to the insertionof an exogenous polynucleotide into a host cell. The exogenouspolynucleotide may be maintained as a non-integrated vector, such as forexample a plasmid, or alternatively may be integrated into the host cellgenome. Examples of transfection techniques include, but are not limitedto, calcium phosphate co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, electroporation and microinjection. Suitablemethods for transfecting host cells can be found in Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory press, and other laboratory textbooks. Nucleic acidcan also be transferred into cells via a delivery mechanism suitable forintroduction of nucleic acid into cells in vivo, such as via aretroviral vector (see e.g., Ferry et al. (1991) Proc. Natl. Acad. Sci.,USA, 88: 8377-8381; and Kay et al. (1992) Human Gene Therapy 3:641-647), an adenoviral vector (see, e.g., Rosenfeld (1992) Cell 68:143-155; and Herz and Gerard (1993) Proc. Natl. Acad. Sci., USA,90:2812-2816), receptor-mediated DNA uptake (see e.g., Wu, and Wu (1988)J. Biol. Chem. 263: 14621; Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320), direct injection of DNA (see,e.g., Acsadi et al. (1991) Nature 332: 815-818; and Wolff et al. (1990)Science 247:1465-1468) or particle bombardment (biolistics) (see e.g.,Cheng et al. (1993) Proc. Natl. Acad. Sci., USA, 90:4455-4459; andZelenin et al. (1993) FEBS Letts. 315: 29-32).

Once expressed, the chimeric lipid binding polypeptides may be purifiedaccording to standard procedures of the art, including, but not limitedto affinity purification, ammonium sulfate precipitation, ion exchangechromatography, or gel electrophoresis.

In some embodiments, a chimeric lipid binding polypeptide may beproduced using a cell free expression system or via solid-state peptidesynthesis.

Modified Lipid Binding Polypeptides

In some embodiments of the invention, a lipid binding polypeptide isprovided that has been modified such that when the polypeptide isincorporated into a bioactive agent delivery particle as describedabove, the modification will increase stability of the particle orconfer targeting ability. In some embodiments, the modification permitsthe lipid binding polypeptides of a particle to stabilize the particle'sdisc shaped structure or conformation. In one embodiment, themodification includes introduction of cysteine residues intoapolipoprotein molecules to permit formation of intramolecular orintermolecular disulfide bonds, e.g., by site-directed mutagenesis. Inanother embodiment, a chemical crosslinking agent is used to formintermolecular links between apolipoprotein molecules to enhancestability of the particles. Intermolecular crosslinking prevents orreduces dissociation of apolipoprotein molecules from the particlesand/or prevents displacement by apolipoprotein molecules within anindividual to whom the particles are administered.

In other embodiments, a lipid binding polypeptide is modified either bychemical derivatization of one or more amino acid residues or by sitedirected mutagenesis, to confer targeting ability to or recognition by acell surface receptor.

Delivery System for Delivery of a Bioactive Agent to an Individual

The invention provides a delivery system for delivery of a bioactiveagent to an individual, comprising bioactive agent delivery particles asdescribed above and a carrier, optionally a pharmaceutically acceptablecarrier. In some embodiments, the delivery system comprises an effectiveamount of the bioactive agent.

As used herein, “individual” refers to any prokaryote or eukaryote towhich one desires to deliver a bioactive agent. In some embodiments, theindividual is a prokaryote such as a bacterium. In other embodiments,the individual is a eukaryote, such as a fungus, a plant, aninvertebrate animal, such as an insect, or a vertebrate animal. In someembodiments, the individual is a vertebrate, such as a human, a nonhumanprimate, an experimental animal, such as a mouse or rat, a pet animal,such as a cat or dog, or a farm animal, such as a horse, sheep, cow, orpig, a bird (i.e., avian individual), or a reptile (i.e., reptilianindividual).

In some embodiments, delivery particles are formulated in a suitablecarrier for administration to an individual. As used herein, “carrier”refers to a relatively inert substance that facilitates administrationof a bioactive agent. For example, a carrier can give form orconsistency to the composition or can act as a diluent.“Pharmaceutically acceptable carriers” refer to carriers that arebiocompatible (i.e., not toxic to the host) and suitable for aparticular route of administration for a pharmacologically effectivesubstance. Suitable pharmaceutically acceptable carriers include but arenot limited to stabilizing agents, wetting and emulsifying agents, saltsfor varying osmolarity, encapsulating agents, buffers, and skinpenetration enhancers. Examples of pharmaceutically acceptable carriersare described in Remington's Pharmaceutical Sciences (Alfonso R.Gennaro, ed., 18th edition, 1990).

As used herein, “effective amount” refers to an amount of a bioactiveagent sufficient to effect desired results. A “therapeutically effectiveamount” or “therapeutic dose” refers to an amount of a bioactive agentsufficient to effect beneficial clinical results, such as for examplereduction or alleviation of a symptom of a disease, reduction oralleviation of a fungal or bacterial infection, etc.

In some embodiments, the delivery system is a pharmaceutical compositioncomprising a bioactive agent delivery particle and a pharmaceuticallyacceptable carrier. In some embodiments, the pharmaceutical compositioncomprises a bioactive agent delivery particle that contains anon-polypeptide bioactive agent and a pharmaceutically acceptablecarrier. In some embodiments, the bioactive agent delivery particle andthe bioactive agent are non-immunogenic when administered to anindividual. Immunogenicity may be measured by methods that are wellknown in the art. For example, immunogenicity may be assessed by anELISA method, for example by probing serum from an individual to whombioactive agent delivery particles have been administered for antibodybinding to equivalent bioactive agent delivery particles bound to animmunosorbent plate.

Methods of Use

The invention provides methods for administering a bioactive agent to anindividual. The methods of the invention include administering adelivery particle as described above that includes a lipid bindingpolypeptide, a lipid bilayer, and a bioactive agent, wherein theinterior of the particle includes hydrophobic surfaces of the lipidbilayer. Optionally, a therapeutically effective amount of the particlesis administered, optionally in a pharmaceutically acceptable carrier.Generally, the particles are disc shaped, with a diameter of about 7 toabout 29 nm, as measured by native pore limiting gradient gelelectrophoresis. Typically, the bioactive agent includes at least onehydrophobic region, which may be integrated into a hydrophobic region ofthe lipid bilayer.

The route of administration may vary according to the nature of thebioactive agent to be administered, the individual, or the condition tobe treated. Where the individual is a mammal, generally administrationis parenteral. Routes of administration include, but are not limited to,intravenous, intramuscular, subcutaneous, transmucosal, nasal,intrathecal, topical, and transdermal. In one embodiment, the particlesare administered as an aerosol. Delivery particles may be formulated ina pharmaceutically acceptable form for administration to an individual,optionally in a pharmaceutically acceptable carrier or excipient. Theinvention provides pharmaceutical compositions in the form of deliveryparticles in a solution for parenteral administration. For preparingsuch compositions, methods well known in the art may be used, and anypharmaceutically acceptable carriers, diluents, excipients, or otheradditives normally used in the art may be used.

The delivery particles of the present invention can be made intopharmaceutical compositions by combination with appropriate medicalcarriers or diluents. For example, the delivery particles can besolubilized in solvents commonly used in the preparation of injectablesolutions, such as for example, physiological saline, water, or aqueousdextrose. Other suitable pharmaceutical carriers and their formulationsare described in Remington's Pharmaceutical Sciences, supra. Suchformulations may be made up in sterile vials containing deliveryparticles and optionally an excipient in a dry powder or lyophilizedpowder form. Prior to use, the physiologically acceptable diluent isadded and the solution withdrawn via syringe for administration to anindividual.

Delivery particles may also be formulated for controlled release. Asused herein, “controlled release” refers to release of a bioactive agentfrom a formulation at a rate that the blood concentration of the agentin an individual is maintained within the therapeutic range for anextended duration, over a time period on the order of hours, days,weeks, or longer. Delivery particles may be formulated in a bioerodibleor nonbioerodible controlled matrix, a number of which are well known inthe art. A controlled release matrix may include a synthetic polymer orcopolymer, for example in the form of a hydrogel. Examples of suchpolymers include polyesters, polyorthoesters, polyanhydrides,polysaccharides, poly(phosphoesters), polyamides, polyurethanes,poly(imidocarbonates) and poly(phosphazenes), andpoly-lactide-co-glycolide (PLGA), a copolymer of poly(lactic acid) andpoly(glycolic acid). Collagen, albumin, and fibrinogen containingmaterials may also be used.

Delivery particles may be administered according to the methodsdescribed herein to treat a number of conditions including, but notlimited to, bacterial infections, fungal infections, disease conditions,metabolic disorders, or as a prophylactic medication, for example toprevent a bacterial or fungal infection (e.g., pre- or post-surgically).Delivery particles may be used, for example, to deliver an anti-tumoragent (e.g., chemotherapeutic agent, radionuclide) to a tumor. In oneembodiment, the lipid binding polypeptide includes a moiety that targetsthe particle to a particular tumor. Delivery particles may also be usedfor administration of nutraceutical substances, i.e., a food or dietarysupplement that provides health benefits. In some embodiments, deliveryparticles are co-administered with other conventional therapies, forexample, as part of a multiple drug “cocktail,” or in combination withone or more orally administered agents, for example, for treatment of afungal infection. Delivery particles may also be administered asinsecticides or herbicides.

In one aspect, the invention provides a method for treating a fungalinfection in an individual. The method includes administering atherapeutically effective amount of an anti-fungal agent in apharmaceutically acceptable carrier to the individual, wherein theanti-fungal agent is incorporated into a particle that includes a lipidbinding polypeptide and a lipid bilayer, wherein the interior of thelipid bilayer is hydrophobic. In one embodiment, the anti-fungal agentis AmB, incorporated into the hydrophobic interior of the lipid bilayer.In some embodiments, the lipid binding polypeptide is a chimeric proteinthat includes a targeting moiety and/or a moiety with biologicalactivity. In one embodiment, the lipid binding polypeptide includes thetargeting moiety yeast α-mating factor peptide. In another embodiment,the lipid binding polypeptide includes the anti-microbial peptidehistatin 5.

In another aspect, the invention provides a method for treating a tumorin an individual. The method includes administering a therapeuticallyeffective amount of a chemotherapeutic agent in bioactive agent deliveryparticles as described above, in a pharmaceutically acceptable carrier.In one embodiment, the chemotherapeutic agent is camptothecin. A lipidbinding polypeptide component of the delivery particles may include atargeting moiety to target the particles to tumor cells. In oneembodiment, vasoactive intestinal peptide (VIP) is attached to the lipidbinding polypeptide. Since breast cancer cells often overexpress the VIPreceptor, in one embodiment, bioactive agent delivery particlescomprising camptothecin and lipid binding polypeptide-VIP chimeras areused in a method of treatment for breast cancer.

Targeting

A delivery particle of the invention may include a targetingfunctionality, for example to target the particles to a particular cellor tissue type, or to the infectious agent itself. In some embodiments,the particle includes a targeting moiety attached to a lipid bindingpolypeptide or lipid component. In some embodiments, the bioactive agentthat is incorporated into the particle has a targeting capability.

In some embodiments, by engineering receptor recognition properties intoa lipid binding polypeptide, such as an apolipoprotein molecule, theparticles can be targeted to a specific cell surface receptor. Forexample, bioactive agent delivery particles may be targeted to aparticular cell type known to harbor a particular type of infectiousagent, for example by modifying the lipid binding polypeptide componentof the particles to render it capable of interacting with a receptor onthe surface of the cell type being targeted.

In one aspect, a receptor-mediated targeting strategy may be used todeliver antileishmanial agents to macrophages, which are the primarysite of infection for protozoal parasites from the genus Leishmania.Examples of such species include Leishmania major, Leishmania donovani,and Leishmania braziliensis. Bioactive agent delivery particlescontaining an antileishmanial agent may be targeted to macrophages byaltering the lipid binding polypeptide component of the particles toconfer recognition by the macrophage endocytic class A scavengerreceptor (SR-A). For example, an apolipoprotein which has beenchemically or genetically modified to interact with SR-A may beincorporated into delivery particles that contain one or more bioactiveagents that are effective against Leishmania species, such as, forexample, AmB, a pentavalent antimonial, and/or hexadecylphosphocholine.Targeting of delivery particles that contain an antileishmanial agentspecifically to macrophages may be used as a means of inhibiting thegrowth and proliferation of Leishmania spp.

In one embodiment an SR-A targeted bioactive agent delivery particlecontaining AmB is administered to an individual in need of treatment fora leishmanial infection. In another embodiment, another antileishmanialagent, such as hexadecylphosphocholine is administered prior,concurrently, or subsequent to treatment with the AmBcontaining-particles.

In some embodiments, targeting is achieved by modifying a lipid bindingpolypeptide, such as an apolipoprotein, to be incorporated into thebioactive agent delivery particle, thereby conferring SR-A bindingability to the particle. In some embodiments, targeting is achieved byaltering the charge density of the lipid binding polypeptide bychemically modifying one or more lysine residues, for example withmalondialdehyde, maleic anhydride, or acetic anhydride at alkaline pH(see, e.g., Goldstein et al. (1979) Proc. Natl. Acad. Sci. 98:241-260).In one embodiment, Apo B-100 or a truncated form thereof, such as theN-terminal 17% of ApoB-100 (residues 1-782 of apoB-17), is modified byreaction with malondialdehyde. In other embodiments, an apolipoproteinmolecule, such as any of the apolipoproteins described herein, may alsobe chemically modified by, for example acetylation or maleylation, andincorporated into a bioactive agent delivery particle containing anantileishmanial agent.

In other embodiments, SR-A binding ability is conferred to a deliveryparticle by modifying the lipid binding polypeptide by site directedmutagenesis to replace one or more positively charged amino acids with aneutral or negatively charged amino acid.

In other embodiments, SR-A recognition is conferred by preparing achimeric lipid binding polypeptide that includes an N- or C-terminalextension having a ligand recognized by SR-A or an amino acid sequencewith a high concentration of negatively charged residues. A negativelycharged polypeptide extension would not be attracted to the lipidsurface of the bioactive agent delivery particle, thereby rendering itmore accessible to the ligand binding site of the receptor.

Methods for Preparing Bioactive Agent Delivery Particles

The invention provides methods for formulating a bioactive agentdelivery particle. In one embodiment, a process is provided thatincludes adding lipid binding polypeptide molecules to a mixture thatincludes bilayer-forming lipids and bioactive agent molecules.

In some embodiments, the lipid-bioactive agent mixture also includes adetergent, such as for example sodium cholate, cholic acid, or octylglucoside, and the process further includes removing the detergent afterthe lipid binding polypeptide has been added. Typically, the detergentis removed by dialysis or gel filtration. In one embodiment, the processincludes combining bilayer-forming lipids and bioactive agent moleculesin a solvent to form a-bioactive agent mixture, drying the mixture toremove the solvent (e.g., under a stream of N₂ and/or bylyophilization), contacting the dried mixture with a solution thatincludes a detergent to form a lipid-bioactive agent-detergent mixture,adding lipid binding polypeptide molecules to this mixture, and thenremoving the detergent.

In some embodiments, the particles are prepared using a microfluidizerprocessor. This procedure employs high pressure, forcing the componentstogether in a reaction chamber.

In some embodiments, the particles are prepared by incubation of asuspension of lipid vesicles containing a bioactive agent in thepresence of a lipid binding polypeptide, such as an apolipoprotein. Inone embodiment, the suspension is sonicated.

In other embodiments, delivery particles are prepared from a pre-formedvesicle dispersion. Lipids, e.g., phospholipids, are hydrated withbuffer and dispersed by agitation or sonication. To the dispersion oflipid bilayer vesicles, solubilized bioactive agent is added in asuitable solvent to form a lipid-bioactive agent complex. In someembodiments, the solvent is volatile or dialyzable for convenientremoval after addition of bioactive agent to the lipid bilayer vesicledispersion. Following further agitation, lipid binding polypeptide isadded and the sample is incubated, mixed by agitation, and/or sonicated.Typically, the vesicles and apolipoprotein are incubated at or near thegel to liquid crystalline phase transition temperature of the particularbilayer forming lipid or mixture of bilayer-forming lipids being used.The phase transition temperature may be determined by calorimetry.

Preferably, a suitable bilayer-forming lipid composition is used suchthat, upon dispersion in aqueous media, the lipid vesicles provide asuitable environment to transition a bioactive agent from a carriersolvent into an aqueous milieu without precipitation or phase separationof the bioactive agent. The pre-formed lipid bilayer vesicles are alsopreferably capable of undergoing lipid binding polypeptide-inducedtransformation to form the delivery particles of the invention. Further,the lipid-bioactive agent complex preferably retains properties of thelipid vesicles that permit transformation into bioactive agent deliveryparticles upon incubation with a lipid binding polypeptide underappropriate conditions. The unique combination of lipidsubstrate-bioactive agent complex organization and lipid bindingpolypeptide properties combine to create a system whereby, underappropriate conditions of pH, ionic strength, temperature, andlipid—bioactive agent—lipid binding polypeptide concentration, a ternarystructural reorganization of these materials occurs wherein stable lipidbinding polypeptide circumscribing lipid bilayers are created with abioactive agent incorporated into the lipid milieu of the bilayer. For adiscussion of the effect of pH, ionic strength and lipid bindingpolypeptide concentration on the ability of lipid binding polypeptidesto induce transformation of different types of phospholipid vesiclesinto disc shaped particles, see Weers et al. (2001) Eur. J. Biochem.268:3728-35.

The particles prepared by any of the above processes may be furtherpurified, for example by dialysis, density gradient centrifugationand/or gel permeation chromatography.

In a preparation method for formation of bioactive agent deliveryparticles, preferably at least about 70, more preferably at least about80, even more preferably at least about 90, even more preferably atleast about 95 percent of the bioactive agent used in the procedure isincorporated into the particles.

The invention provides bioactive agent delivery particles prepared byany of the above methods. In one embodiment, the invention provides apharmaceutical composition comprising a delivery particle prepared byany of the above methods and a pharmaceutically acceptable carrier.

Storage and Stability

Particles of the invention are stable for long periods of time under avariety of conditions (see, for example, FIG. 5). Particles, orcompositions comprising particles of the invention, may be stored atroom temperature, refrigerated (e.g., about 4° C.), or frozen (e.g.,about −20° C. to about −80° C.). They may be stored in solution or dried(e.g., lyophilized). Bioactive agent delivery particles may be stored ina lyophilized state under inert atmosphere, frozen, or in solution at 4°C. Particles may be stored in a liquid medium, such as a buffer (e.g.,phosphate or other suitable buffer), or in a carrier, such as forexample a pharmaceutically acceptable carrier, for use in methods ofadministration of a bioactive agent to an individual. Alternatively,particles may be stored in a dried, lyophilized form and thenreconstituted in liquid medium prior to use.

Kits

The reagents and particles described herein can be packaged in kit form.In one aspect, the invention provides a kit that includes deliveryparticles and/or reagents useful for preparing delivery particles, insuitable packaging. Kits of the invention include any of the following,separately or in combination: lipid binding polypeptides (e.g.,apolipoproteins), phospholipids, bioactive agents, vectors, reagents,enzymes, host cells and/or growth medium for cloning and/or expressionof recombinant lipid binding polypeptides (e.g., recombinantapolipoproteins) and/or lipid binding polypeptide chimeras (e.g.,apolipoprotein chimeras), and reagents and/or pharmaceuticallyacceptable carriers for formulating delivery particles foradministration to an individual.

Each reagent or formulation is supplied in a solid form, liquid buffer,or pharmaceutically acceptable carrier that is suitable for inventorystorage, or optionally for exchange or addition into a reaction,culture, or injectable medium. Suitable packaging is provided. As usedherein, “packaging” refers to a solid matrix or material customarilyused in a system and capable of holding within fixed limits one or moreof the reagents or components (e.g., delivery particles) for use in amethod for delivery of a bioactive agent or one or more reagents forpreparing or formulating delivery particles (e.g., apolipoproteinmolecules, phospholipids, bioactive agents). Such materials include, butare not limited to, glass and plastic (e.g., polyethylene,polypropylene, and polycarbonate) bottles, vials, paper, plastic, andplastic-foil laminated envelopes, and the like.

A kit may optionally provide additional components that are useful inthe methods and formulation procedures of the invention, such asbuffers, reacting surfaces, or means of purifying delivery particles.

In addition, the kits optionally include labeling and/or instructionalor interpretive materials providing directions (i.e., protocols) for thepractice of the methods of this invention, such as preparation,formulation and/or use of delivery particles. While the instructionalmaterials typically comprise written or printed materials they are notlimited to these formats. Any medium capable of storing suchinstructions and communicating them to an end user is contemplated bythis invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses toInternet sites that provide such instructional materials.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLES Example 1 Preparation and Characterization ofApoA-I-Phospholipid-Amphotericin B Particles Preparation of RecombinantApoA-I

Recombinant Apo-A-I was prepared as described in Ryan et al. (2003)Protein Expression and Purification 27:98-103, and was used to prepareApo-A-1-phospholipid-AmB particles, as described below.

Preparation of ApoA-I-Phospholipid-AmB Particles

ApoA-I-phospholipid-AmB particles were prepared as follows:

A 7:3 molar ratio of dimyristoylphosphatidylcholine (DMPC) anddimyristoylphosphatidylglycerol (DMPG) were dissolved inchloroform:methanol (3:1, v/v). To 10 mg of the DMPC/DMPG mixture, 0.25ml of AmB (2 mg/ml; solubilized in acidified chloroform:methanol (3:1,v/v)) was added. The mixture was dried under a stream of N₂ gas tocreate a thin film on the vessel wall. The dried sample was thensubjected to lyophilization for sixteen hours to remove traces ofsolvent.

The dried lipid mixture was resuspended in 0.5 ml Tris-Saline buffer (10mM Tris base 150 mM NaCl, pH 8), and the mixture was vortexed for 30seconds.

To the resuspended lipid mixture, 0.5 ml of 22 mM sodium cholate wasadded to the mixture and vortexed for 3 minutes. This mixture wasincubated at 37° C. with vortexing every 10 minutes for 1.25 hours oruntil the mixture was clear. To the cleared solution, 2 ml of isolatedrecombinant ApoA-I, prepared as described in Example 1, was added at aconcentration of 1.5 mg/ml, and the mixture was incubated at 37° C. foran additional 1 hour. To remove sodium cholate, the sample was subjectedto dialysis against 4 liters of Tris-Saline at 4° C. for 72 hours with achange of dialysis buffer every 24 hours.

The sample was further purified by density gradient ultracentrifugation.The solution was adjusted to a density of 1.30 g/ml by the addition ofsolid KBr in 1.5 ml. The sample was transferred to a 3 ml centrifugetube, overlayered with saline and centrifuged at 275,000×g for 3 hoursin a Beckman L7-55 centrifuge.

Particle Stability

The particles prepared according to this procedure were stable for morethan 3 months in lyophilized form.

Characterization of Particles

UV/visible scans were performed for ApoA-I-phospholipid particles,prepared as described above but without the addition of AmB, and werecompared with scans for the AmB-containing particles. FIG. 1 shows thescan for particles that do not include AmB. The only peak observed was aprotein peak at around 280 nm. FIG. 2 shows the scan for AmB-containingparticles prepared as described above. In addition to the peak at around280 nm, a number of additional peaks were observed in the 300-400 nmregion of the spectrum, confirming the presence of AmB. Free AmB isinsoluble in aqueous media and has different spectral properties thanobserved in FIG. 2. Madden et al. (1990) Chemistry and Physics ofLipids, 52:189-98.

Characterization studies revealed that the ApoA-I, phospholipid, and AmBmigrate as a discrete particle population when subjected to densitygradient ultracentrifugation (FIG. 3). The complexes float to acharacteristic density in the gradient that is dependent upon theprotein/lipid ratio in the particles.

Further, gradient gel electrophoresis under non-denaturing conditionsrevealed that the major complex generated is of uniform size, displayinga Stokes' diameter of 8.5 nm (FIG. 4). Analysis of the isolatedparticles revealed that significant deviation from the original molarratios of AmB, phospholipid, and apolipoprotein did not occur.

Example 2 Antifungal Activity of AmB Containing Bioactive Agent DeliveryParticles Against Saccharomyces cerevisiae

ApoA-I-DMPC/DMPG-AmB particles were prepared as described in Example 1and used to determine antifungal activity of the complexes. Cultures ofS. cerevisiae were grown in YPD medium in the presence of varyingamounts of ApoA-1-DMPC/DMPG-AmB particles (0-25 μg AmB/ml). The cultureswere grown for 16 hours at 30° C., and the extent of culture growthmonitored spectrophotometrically. As shown in FIG. 8, the AmB-containingparticles were extremely effective in inhibiting fungal growth in adose-dependent manner.

Example 3 Long Term Stability of Bioactive Agent Delivery Particles

Recombinant ApoE3NT-terminal domain (ApoE3NT) was prepared as in Fisheret al. (1997) Biochem Cell Biol 75:45-53. ApoE3NT-AmB-containingparticles were prepared via the cholate dialysis method described inExample 1, and used to assess long-term stability.

FIG. 5 shows a native PAGE 4-20% gradient slab gel of particles storedin phosphate buffer at 4° C. (lane 1), stored in phosphate buffer at−20° C. (lane 2), or frozen in phosphate buffer at −80° C., lyophilized,and redissolved in H₂O prior to analysis. The size and mobility of theAmB-containing particles were unaffected by freezing and thawing, or bylyophilization and resolubilization, indicating that the particlesretained their integrity under these conditions. These are importantparameters with regard to scale up and long-term storage of AmB deliveryparticles.

Example 4 Preparation of AmB-Containing Bioactive Agent DeliveryParticles with POPC

ApoA-I-POPC particles were prepared using the cholate dialysis methoddescribed in Example 1. A native PAGE gradient gel analysis ofApoA-I-POPC particles is shown in FIG. 4. Particles without AmB areshown in lane 1 and particles with AmB are shown in lane 2. The gelindicates that incorporation of AmB into the particles does not altertheir size. However, the gel indicates that the POPC containingparticles are a different size than DMPC/DMPG particles, shown in lane3.

Example 5 Preparation of AmB-Containing Particles with a MicrofluidizerProcessor

ApoA-I, AmB, egg PC, DPPG, and cholesterol were combined in amicrofluidizer sample holder and passed through the reaction chamber ofmicrofluidizer processor at 18,000 psi. The resultant solution wascollected and characterized in terms of particle formation,incorporation of hydrophobic substances, size, and stability.AmB-containing particles of about 16 nm diameter were obtained, whichwere stable to lyophilization and aqueous solvent reconstitution.

Example 6 Preparation of AmB-Containing Particles from PhospholipidVesicles

A suspension of AmB-containing phospholipid vesicles was prepared byadding an aliquot of a 20-40 mg/ml solution of AmB in DMSO,corresponding to 2.5 mg AmB, to a preformed phospholipid aqueousdispersion containing a molar ratio of 7:3 DMPC:DMPG. The vesicles wereincubated at the gel to liquid phase transition temperature of thephospholipids (about 24° C.). Addition of 4 mg apolipoprotein led to atime-dependent decrease in sample turbidity, consistent with formationof AmB-containing bioactive agent delivery particles. Full sampleclarity was achieved by mild bath sonication at 21-25° C. for 1-20minutes or in 4-16 hours without sonication at 24° C. The resultingparticles exhibited >90% AmB incorporation efficiency, i.e., thepercentage of AmB starting material that is recovered in deliveryparticles, and no material was lost upon filtration, centrifugation, ordialysis. Other tests revealed that similar results can be achieved withAmB concentration adjusted to as high as 5 mg/10 mg phospholipid. Thisprocedure worked equally well with any of five apolipoproteins tested(ApoA-I, ApoE3NT, Bombyx mori ApoIII, and a variant form of human ApoA-Ithat includes a C-terminal extension including the antifungal peptide,Histatin 5, and a variant form of human ApoA-I that includes aC-terminal extension including the S. cerevisiae α-mating factorpeptide).

Density gradient ultracentrifugation of ApoA-I or ApoE3NT containingparticles revealed a single population of particles that floated to acharacteristic density in the range of 1.21 g/ml, consistent withformation of lipid-protein complexes. Characterization of the fractionsobtained following density gradient ultracentrifugation revealed thatphospholipid, AmB, and apolipoprotein migrated to the same position inthe gradient, consistent with formation of AmB-containing particles.

Comparison of the relative migration of ApoA-I-AmB-containing particleswith known standards on native PAGE indicated that over 90% of theparticles had a Stokes' diameter of approximately 8.5 nm. This value issimilar to particles generated in the absence of AmB, indicating thataddition of this bioactive agent did not significantly alter the sizedistribution of the particles.

As a measure of the overall stability of the ApoA-I-AmB-containingbioactive agent delivery particles, the particles were frozen at −20° C.or lyophilized. Freezing/thawing had no effect on the size distributionof the particles. Likewise, subjecting the particles to lyophilizationand re-dissolving in H₂O did not affect the size distribution or sampleappearance.

These data strongly suggest that AmB, phospholipids, and apolipoproteincombined to form a homogeneous population of bioactive agent deliveryparticles in which AmB is fully integrated into the bilayer portion ofthe particle. Spectrophotometric analysis of the AmB-containingparticles revealed a characteristic set of peaks in the visible rangethat are consistent with AmB solubilization in the bioactive agentdelivery particle.

Example 7 Comparison of AmB-Containing Particles Prepared as in Example6 with Particles Prepared by an Alternate Procedure

Incorporation of bioactive agent into bioactive agent delivery particlesusing the method described in Example 6 was compared with incorporationinto “neo-HDL” particles prepared according to Shouten et al. (1993)Molecular Pharmacology 44:486-492, as follows: Three mg of egg yolkphosphatidylcholine, 0.9 mg cholesterol, and 1.5 mg AmB, dissolved inchloroform, were mixed in a 20 ml glass vial, and the solvent wasevaporated under a stream of nitrogen. Ten ml of sonication buffer (10mM Tris HCl, pH 8.0, 100 mM KCl, 1 mM EDTA, and 0.025% NaN₃), degassedand saturated with nitrogen, were added and the contents of the vialsonicated with a Macrotip (14 μm average output) under a stream ofnitrogen. The temperature was maintained above 41° C. and below 50° C.The sonication was stopped after 60 minutes, and the temperatureadjusted to 42° C. Sonication was continued and 20 mg of ApoA-I,dissolved in 2 ml of 4M urea, was added in ten equal portions over aperiod of 10 minutes. After all of the protein was added, sonication wascontinued for 30 min at 42° C.

The sonication mixture was then centrifuged for 3 minutes to removelarge particles and insoluble material and the supernatant analyzed byUV/Visible spectrophotometry to assess the amount of amphotericin Bsolubilized in the product particles. It was noted that the solution wasslightly opaque. The sample was scanned from 250 nm to 500 nm. Forcomparison, AmB-containing particles prepared by the procedure describedin Example 6 were examined. The results are shown in FIG. 12. The regionof the spectrum arising from AmB (300-500 nm) is quite distinct betweenthe two samples. Whereas AmB-containing bioactive agent deliveryparticles generated using the protocol described in Example 6 had strongcharacteristic absorbance maxima that indicate solubilization andincorporation of AmB into the particles (Madden et al., supra) (FIG.12B), the sample prepared according to Schouten et al. did not give riseto these characteristic spectral maxima (FIG. 12A). Indeed, the spectrumobtained is very similar to that reported by Madden et al., supra, foran aqueous dispersion of AmB in the absence of lipid. Thus, AmB was notincorporated into lipid particles using this procedure, whereas theprocedure described in Example 6 resulted in significant AmBincorporation.

Example 8 Comparison of Anti-Fungal Activity of AmB-Containing BioactiveAgent Delivery Particles with Liposomal AmB Formulation

Anti-fungal activity of ApoA-I-AmB particles, prepared as in Example 6,and a commercial liposomal formulation of AmB, AmBisome®, were comparedwith respect to their ability to inhibit the growth of the yeast, S.cerevisiae. The data in FIG. 10 show that Apo-A-1-AmB bioactive agentdelivery particles more effectively inhibited S. cerevisiae growth thanthe same amount of AmB formulated as AmBisome®. Apo-A-1-AmB bioactiveagent delivery particles achieved 90% growth inhibition at 1 μg/ml,whereas this level of inhibition required 25 μg/ml AmBisome®.

Anti-fungal activity of ApoA-I-AmB particles and AmBisome® were alsocompared against two species of pathogenic fungi, Candida albicans (C.albicans) and Aspergillus fumigatus (A. fumigatus), in microtiter brothwhole-cell assays. As a control, particles without AmB were also tested.The results are shown in Table 1.

TABLE 1 Amphotericin B Inhibition of Pathogenic Fungal Growth ED₉₀(μg/ml) Delivery Control particles particles Organism AmBisome with AmBwithout AmB Candida albicans 0.8 0.1 No inhibition Aspergillus fumigatus1.6 0.2 No inhibition

The results obtained revealed that AmB-containing bioactive agentdelivery particles were effective against both pathogenic fungalspecies, at a lower concentration than AmBisome®. Control particleslacking AmB were not effective. AmB-containing bioactive agent deliveryparticles exhibited an ED₉₀ (concentration at which 90% growthinhibition is observed) for C. albicans at a concentration of 0.1 μg/ml,whereas 0.8 μg/ml AmBisome® was required to achieve the same level ofgrowth inhibition. For A. fumigatus, AmB-containing bioactive agentdelivery particles inhibited 90% of fungal growth at a concentration of0.2 μg/ml, whereas 1.6 μg/ml AmBisome® was required to achieve the sameeffect.

In another experiment, AmB-containing bioactive agent delivery particlescontaining apolipophorin III as the lipid-binding polypeptide werecompared with AmBisome® for their ability to inhibit growth of threespecies of pathogenic fungi, C. albicans, A. fumigatus, and Cryptococcusneoformans (C. neoformans). The data are shown in Table 2.

TABLE 2 Amphotericin B Inhibition of Pathogenic Fungal Growth ED₉₀(μg/ml) Delivery Control particles particles Organism AmBisome with AmBwithout AmB Candida albicans 0.4 0.03 No inhibition Aspergillusfumigatus 2.5 0.1 No inhibition Cryptococcus neoformans 0.31 0.06 Noinhibition

AmB-containing bioactive agent delivery particles inhibited 90% of C.albicans growth at 0.03 μg/ml. A corresponding ED₉₀ of 0.4 μg/ml wasobtained with AmBisome®. In the case of A. fumigatus, AmB-containingbioactive agent delivery particles inhibited 90% of fungal growth at 0.1μg/ml, whereas a concentration of 2.5 μg/ml AmBisome® was required toachieve the same effect. In a similar manner, AmB-containing particleswere effective at inhibiting C. neoformans growth at a five-fold lowerAmB concentration than AmBisome®.

All samples tested were soluble in the RPMI media used for theexperiments and no precipitation or interference was observed in any ofthe samples tested against any of the fungal species. These data suggestthat a formulation of AmB in the bioactive agent delivery particles ofthe invention has more potent anti-fungal activity than a liposomalformulation.

Example 9 Incorporation of Camptothecin into Bioactive Agent DeliveryParticles

Camptothecin-containing bioactive agent delivery particles were preparedas follows: A 7:3 molar ratio of DMPC:DMPG (5 mg total) was dispersed inbuffer (20 mM sodium phosphate, pH 7.0) by vortexing for 1 minute togenerate a dispersion of phospholipid bilayer vesicles. Ten microlitersof a 10 mg/ml solution of camptothecin in DMSO was added to thephospholipid bilayer dispersion. Two mg of recombinant humanapolipoprotein A-I (0.5 ml of a 4 mg/ml solution in 20 mM sodiumphosphate, pH 7.0) was then added, and the sample was then subjected tosonication. The clarified sample was then centrifuged at 13,000×g for 3minutes and the supernatant recovered and stored at 4° C.

A fluorescence spectrum of the camptothecin-containing particles, incomparison with sodium dodecyl sulfate (SDS) solubilized camptothecin,is shown in FIG. 11. Fluorescence measurements were obtained on a PerkinElmer LS 50B luminescence spectrometer at an excitation wavelength of360 nm with emission monitored from 400 to 600 nm. The blue shift influorescence emission maximum elicited by camptothecin in SDS micelles(FIG. 11A) compared to camptothecin incorporated into bioactive agentdelivery particles (FIG. 11B) suggests that the drug localizes to a morehydrophobic environment in the micelles versus the delivery particles.

Example 10 Freeze Fracture Electron Microscopy of AmB-ContainingBioactive Agent Delivery Particles

A preparation of AmB-containing bioactive agent delivery particles wasprepared for freeze fracture electron microscopy as follows: A sample ofDMPC:DMPG (7:3 molar ratio) AmB bioactive agent delivery particles (3mg/ml protein), prepared as in Example 6, was quenched using a sandwichtechnique, and liquid nitrogen cooled propane. The cryo-fixed sample wasstored in liquid nitrogen for less than 2 hours prior to processing. Thefracturing process was carried out in JOEL JED-900 freeze-etchingequipment and the exposed fracture planes were shadowed with Pt for 30seconds at an angle of 25-35 degrees, and with carbon for 35 seconds (2kV/60-80 mA, 1×10⁻⁵ Torr). The replicas produced in this way werecleaned with concentrated fuming HNO₃ for 24 hours followed by repeatedagitation with fresh chloroform/methanol (1:1 by volume) at least 5times. The replicas cleaned in this way were examined on a JOEL 100 CXor a Philips CM 10 electron microscope.

An electron micrograph obtained from freeze fracture of AmB-containingparticles as described above is shown in FIG. 9. Electron micrographstaken from several freeze-fracture preparations indicate the presence ofsmall protein-lipid complexes in high concentration. The apparentdiameters range from about 20-60 nm with high frequency around 40 nm.The apparent diameter of particles as observed by freeze fractureelectron microscopy is larger than values obtained by native porelimiting gradient gel electrophoresis. The difference may be due to theeffect of sample handling or the staining procedure used to visualizethe particles by electron microscopy.

The substantially spherical complexes do not display concave or convexfracture faces (shadow in front and behind the structure, respectively),as are characteristic for liposomes. Further, no evidence for micellarstructures was observed.

Example 11 In Vivo Assessment of Anti-Fungal Activity of AmB-ContainingBioactive Agent Delivery Particles in Immunocompetent Mice

In vivo anti-fungal activity of AmB-containing bioactive agent deliveryparticles is assessed as follows:

Animals

Six to eight-week-old female BALB/c mice (20-25 g) are housed andmaintained under standard laboratory conditions.

Toxicity Study

Groups of three mice each receive a dose (e.g., 1, 2, 5, 10, or 15 mg/kgAmB) in AmB-containing bioactive agent delivery particles, or controlparticles without AmB, in saline buffered to pH 7.4 with 10 mM sodiumphosphate. A single dose is administered as a 0.1 ml volumeintraperitoneally. Preliminary studies have indicated that the bioactiveagent delivery particles are fully soluble under these conditions.

Following injection, the mice are observed for any general reaction, forexample, abnormal movement or posture, difficulty in breathing, ruffledfur, or inability to obtain food or drink. Observation for abnormalityor mortality begins immediately after administration and continues twicedaily for seven days. Body weight is recorded daily for the same period.

Blood is collected from mice prior to euthanization. The blood isassayed for liver enzymes such as lactate dehydrogenase to assess thedegree of liver specific damage

Efficacy of AmB-Containing Bioactive Agent Delivery Particles inTreatment of Systemic Cryptococcus

The therapeutic range of AmB-containing particles is determined andcompared with AmBisome® as follows:

A clinical isolate of C. neoformans that is susceptible to AmB iscultured and prepared as an inoculum for infection at a concentration of2×10⁶ conidia/ml. Each mouse receives an inoculum of 1×10⁵ conidia in0.05 ml of normal saline intracranially under general anesthesia.

Anti-fungal agents are administered intraperitoneally in 0.1 ml volumesdaily for 5 days, starting 2 hours post-infection. The dosage of AmBused is determined based on the toxicity studies described above. Onetreatment group of mice receives AmBisome®, one treatment group receivesAmB-containing bioactive agent delivery particles, and a control groupreceives no therapy.

Infected mice are monitored twice daily and any signs of illness ormortality is recorded for up to 28 days. Body weight is recorded dailyfor the same time period. Moribund animals that fail to move normally ortake food or drink are euthanized. Based on the outcome of thesestudies, a second set of studies is performed to verify and reproducethe findings. The AmB dose employed, as well as the number of mice inthe control and treatment groups, may be adjusted to reflect knowledgegained from the previous experiment.

Determination of Tissue Fungal Burden

Mice are sacrificed one day after the last day of treatment. The kidneysand brains are removed aseptically and weighed. Tissues are homogenizedand serially diluted in normal saline. The homogenates are cultured for48 hours on PDA (potato dextrose agar) plates to determine the colonyforming units (CFU). Fungal burden of CFU/gram of tissue is determined.

Statistical Analyses

Differences in survival and mean CFUs in kidney or brain are comparedusing statistical tests as appropriate.

Pharmacokinetic Study

Blood, liver, kidney, ling, and cerebrospinal fluid samples arecollected from infected mice at time points of 10 minutes, 2, 8, and 24hours after intravenous injection of AmB bioactive agent deliveryparticles or AmBisome® at 0.8 and 2.0 mg/kg doses. While mice are undergeneral anesthesia, whole blood is collected from axillary vessels. Athoracotomoy is performed, and tissue samples perfused with normalsaline and then removed surgically. Tissues are homogenized withmethanol containing 1-amino-4-nitronaphthalene. Serum and thesupernatants of tissue homogenates are preserved until analysis. Theconcentration of AmB in each sample is determined by high-performanceliquid chromatography (HPLC), as described in Granich et al. (1986)Antimicrob. Agents Chemother. 29:584-88. Briefly, serum samples (0.1 ml)are combined with 1.0 ml methanol containing 1.0 mg of an internalstandard, 1-amino-4-nitronaphthalene, per ml and mixed by vortexing.After centrifugation, the supernatant is dried under reduced pressurefollowed by redissolving with 0.2 ml of methanol for injection onto aHPLC column (C₁₈ reverse phase). Weighed wet tissue samples arehomogenized in 10 volumes of methanol containing 5.0 mg internalstandard per ml with a glass homogenizer and centrifuged. The mobilephase is a mixture of acetonitrile and 10 mM sodium acetate buffer (pH4.0; 11:17 (vol/vol)), at a flow rate of 1.0 ml/min. The concentrationof AmB is determined by the ratio of the peak height of AmB to that ofthe internal standard.

Example 12 Targeting of Camptothecin-Containing Bioactive Agent DeliveryParticles to Tumor Cells

Bioactive agent delivery agent particles are prepared with a VIPtargeting moiety attached to the lipid binding polypeptide component.

The lipid binding polypeptide component of the camptothecin-containingparticles may be generated in recombinant form in Escherichia coli (E.coli) that have been transformed with a plasmid vector harboring thecoding sequence of the lipid binding polypeptide. For example,recombinant human ApoA-I may be employed. E. coli cells harboring anApoA-I expression plasmid are cultured in media at 37° C. When theoptical density of the culture at 600 nm reaches 0.6, ApoA-I synthesisis induced by the addition of isopropylthiogalactoside (0.5 mM finalconcentration). After a further 3 hours of culture, the bacteria arepelleted by centrifugation and disrupted by sonication. The cell lysateis centrifuged at 20,000×g for 30 min at 4° C. and apoA-I isolated fromthe supernatant fraction.

A recombinant lipid binding polypeptide chimera is produced byengineering ApoA-I to include an N-terminal and/or C-terminal peptideextension that corresponds to the 28 amino acid neuropeptide, vasoactiveintestinal peptide (VIP). ApoA-I-VIP chimeras may be employed to createbioactive agent delivery particles comprised of phospholipid,camptothecin and ApooA-I-VIP chimera.

For example, an ApoA-I-VIP chimera may be constructed by synthesizingcomplementary oligonucleotide primers corresponding to the codingsequence of the VIP sequence possessing terminal Hind III and Xba Isites. The oligonucleotides (˜100 base pairs) are annealed to generatedouble stranded DNA with the desired “sticky ends” and subcloned intothe ApoA-I coding sequence-containing plasmid vector that hasappropriately placed Hind III and Xba I restriction enzyme sites.Following ligation, transformation and screening for a positive chimeraconstruct, the plasmid DNA is isolated and subject to automated dideoxychain termination sequence analysis. Following confirmation that thesequence corresponds to that predicted for the desired chimera,production of recombinant ApoA-I-VIP chimera is performed in E. coli, asdescribed above for wild type ApoA-I. Purified recombinant chimera isthen evaluated by gel electrophoresis, mass spectrometry and for itsability to generate bioactive agent delivery particles of the inventionin a manner similar to wild type ApoA-I, as described in Example 8.

ApoA-I-VIP chimera-camptothecin-containing bioactive agent deliveryparticles may be used in breast cancer cell growth inhibition studies tomeasure the extent of lipid particle targeting. For example, the humanbreast cancer cell line MCF-7 is obtained from the American Type CultureCollection and maintained at 37° C. in a humidified 5% CO₂ incubator asmonolayer cultures in modified Eagle's media supplemented with 10% fetalbovine serum and the antibiotics penicillin and streptomycin. Isolatedwild type ApoA-I or ApoA-I-VIP chimera is radioiodinated andincorporated into camptothecin-containing bioactive agent deliveryparticles of the invention and incubated with the cells. Cell-associatedradioactivity is determined after incubation of labeledcamptothecin-containing bioactive agent delivery particles with culturedMCF-7 cells at 4° C. The ability of VIP to compete for binding ofApoA-I-VIP chimera or bioactive agent delivery particle-associatedApoA-I-VIP chimera to MCF cells is determined in competition bindingassays. Cell binding data is evaluated by Scatchard analysis. The extentof MCF-7 cell internalization of ApoA-I-VIP chimera bioactive agentdelivery particles is evaluated in incubations with radioiodinatedApoA-I-VIP chimera-containing bioactive agent delivery particles at 37°C. After incubation and washing, trichloroacetic acid solubleradioactivity is determined, providing a measure of lipid bindingpolypeptide degradation.

Growth inhibition and cytotoxicity studies with different bioactiveagent delivery particles are assessed by clonogenic assay. Exponentiallygrowing cells are resuspended in media and cell number determined usingan electronic counter. Alternatively, camptothecin-ApoA-I-VIP chimerabioactive agent delivery particle inhibition of MCF-7 clonal growth maybe evaluated on the basis of reduced ³⁵S-methionine uptake. Aliquots ofcells are inoculated in triplicate into culture dishes. Afterincubation, specific lipid particles are added from a stock solution tothe dishes to achieve final concentrations of 0, 0.1, 1, 5, 10, 50, 100,and 250 nM camptothecin. After specific time intervals ranging from 0 to72 hours, medium is removed by aspiration and fresh medium added. Thepercentage survival at each drug concentration with different exposuretimes is determined from the ratio of the number of Trypan Blueexcluding cells and compared to results obtained with control particleslacking camptothecin.

Although the foregoing invention has been described in some detail byway of illustration and examples for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced without departingfrom the spirit and scope of the invention. Therefore, the descriptionshould not be construed as limiting the scope of the invention, which isdelineated by the appended claims.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes andto the same extent as if each individual publication, patent, or patentapplication were specifically and individually indicated to be soincorporated by reference.

We claim: 1-63. (canceled)
 64. A pharmaceutical composition comprising abioactive agent delivery particle, wherein said bioactive agent deliveryparticle comprises: a. ApoA-I, b. a lipid bilayer, c. a bioactive agent,and d. a pharmaceutically acceptable carrier; wherein said lipid bilayercomprises at least one phospholipid and the interior of the lipidbilayer comprises a hydrophobic region and said bioactive agent isincorporated into said lipid bilayer; wherein said bioactive agent is abioactive lipid; and wherein said particle does not comprise an aqueouscore, is disc-shaped with the hydrophobic edge of the lipid bilayercircumscribed by ApoA-I at the periphery of the particle and remainsdisc shaped in aqueous solution.
 65. The pharmaceutical composition ofclaim 64, wherein said lipid bilayer comprises two or more phospholipidsselected from the group consisting of DMPC, DMPG, POPC, DPPC, DPPS,cardiolipin, DPPG, DSPG, phosphatidylcholine, phosphatidylinositol,phosphatidic acid, sphingomyelin, and cationic phospholipids.
 66. Thepharmaceutical composition of claim 65, wherein said lipid bilayercomprises sphingomyelin and DPPG.
 67. The pharmaceutical composition ofclaim 66, wherein the molar ratio of DPPG to sphingomyelin is at leastabout 1:50.
 68. The pharmaceutical composition of claim 64, wherein saidbioactive agent is sphingomyelin.
 69. The pharmaceutical composition ofclaim 66, wherein said bioactive agent is sphingomyelin.
 70. Thepharmaceutical composition of claim 65, wherein said lipid bilayercomprises sphingomyelin and POPC.
 71. The pharmaceutical composition ofclaim 67, wherein said bioactive agent is sphingomyelin.
 72. Thepharmaceutical composition of claim 64, wherein said bioactive agent isa phosphatidylcholine.
 73. The pharmaceutical composition of claim 72,wherein said bioactive agent is POPC.
 74. The pharmaceutical compositionof claim 64, wherein said pharmaceutically acceptable carrier comprisesa solvent.
 75. The pharmaceutical composition of claim 66, wherein saidpharmaceutically acceptable carrier comprises a solvent.
 76. Thepharmaceutical composition of claim 74, wherein said solvent is selectedfrom the group consisting of physiological saline, water, and aqueousdextrose.
 77. The pharmaceutical composition of claim 75, wherein saidsolvent is selected from the group consisting of physiological saline,water, and aqueous dextrose.
 78. The pharmaceutical composition of claim64 formulated as a dry powder that is diluted with a solvent prior toadministration to a patient in need thereof.
 79. The pharmaceuticalcomposition of claim 66 formulated as a dry powder that is diluted witha solvent prior to administration to a patient in need thereof.
 80. Thepharmaceutical composition of claim 64 formulated for controlledrelease.
 81. The pharmaceutical composition of claim 66 formulated forcontrolled release.
 82. The pharmaceutical composition of claim 80,wherein said controlled release formulation comprises a syntheticpolymer.
 83. The pharmaceutical composition of claim 82, wherein saidsynthetic polymer is selected from the group comprising polyesters,polyorthoesters, polyanhydrides, polysaccharides, poly(phosphoesters),polyamides, polyurethanes, poly(imidocarbonates), poly(phosphazenes),PLGA, a copolymer of poly(lactic acid) and poly(glycolic acid).
 84. Thepharmaceutical composition of claim 80 wherein said controlled releaseformulation comprises a matrix selected from the group consisting ofcollagen, albumin, and fibrinogen.
 85. The pharmaceutical composition ofclaim 81, wherein said controlled release formulation comprises asynthetic polymer.
 86. The pharmaceutical composition of claim 85,wherein said synthetic polymer is selected from the group comprisingpolyesters, polyorthoesters, polyanhydrides, polysaccharides,poly(phosphoesters), polyamides, polyurethanes, poly(imidocarbonates),poly(phosphazenes), PLGA, a copolymer of poly(lactic acid) andpoly(glycolic acid).
 87. The pharmaceutical composition of claim 81,wherein said controlled release formulation comprises a matrix selectedfrom the group consisting of collagen, albumin, and fibrinogen.
 88. Amethod of treating a condition in a patient comprising administering thepharmaceutical composition of claim 64 to said patient.
 89. A method oftreating a condition in a patient comprising administering thepharmaceutical composition of claim 66 to said patient.
 90. A method oftreating a condition in a patient comprising administering thepharmaceutical composition of claim 70 to said patient.
 91. A method oftreating a condition in a patient comprising administering thepharmaceutical composition of claim 71 to said patient.
 92. A method oftreating a condition in a patient comprising administering thepharmaceutical composition of claim 72 to said patient.
 93. A method oftreating a condition in a patient comprising administering thepharmaceutical composition of claim 73 to said patient.
 94. The methodof claim 88, wherein said condition is selected from the groupconsisting of bacterial infections, fungal infections, metabolicdisorders, and tumors.
 95. The method of claim 94, wherein saidcondition is a metabolic disorder.
 96. A method of treating a patientwith a prophylactic medication comprising administering thepharmaceutical composition of claim 64 to said patient.
 97. A method oftreating a patient with a prophylactic medication comprisingadministering the pharmaceutical composition of claim 66 to saidpatient.
 98. A method of delivering a nutraceutical, comprisingadministering the pharmaceutical composition of claim 64, wherein saidbioactive agent is said nutraceutical.
 99. The method of claim 97,wherein said pharmaceutical composition is coadministered with aconventional therapy.